Taurai Tasara

16S rRNA gene based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification

BackgroundE. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated.ResultsBy phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii species. The newly developed 16S rRNA gene targeting PCR system allows identification of E. sakazakii strains from both lineages. The assays ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other microorganisms was shown on 75 target and non-target strains.ConclusionBy this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify E. sakazakii isolates from both phylogenetic distinct lines within the E. sakazakii species. The impact of this second newly described phylogenetic line within the E. sakazakii species in view of clinical and food safety aspects need further investigation.

Role of Cold Shock Proteins in Growth of Listeria monocytogenes under Cold and Osmotic Stress Conditions

ABSTRACT The gram-positive bacterium Listeria monocytogenes is a food-borne pathogen of both public health and food safety significance. It possesses three small, highly homologous protein members of the cold shock protein (Csp) family. We used gene expression analysis and a set of mutants with single, double, and triple deletions of the csp genes to evaluate the roles of CspA, CspB, and CspD in the cold and osmotic (NaCl) stress adaptation responses of L. monocytogenes. All three Csps are dispensable for growth at optimal temperature (37°C). These proteins are, however, required for efficient cold and osmotic stress tolerance of this bacterium. The hierarchies of their functional importance differ, depending on the environmental stress conditions: CspA>CspD>CspB in response to cold stress versus CspD>CspA/CspB in response to NaCl salt osmotic stress. The fact that Csps are promoting L. monocytogenes adaptation against both cold and NaCl stress has significant implications in view of practical food microbial control measures. The combined or sequential exposure of L. monocytogenes cells to these two stresses in food environments might inadvertently induce cross-protection responses.

Serotypes, intimin variants and other virulence factors of eae positive Escherichia coli strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae-η2)

BackgroundEnteropathogenic Escherichia coli (EPEC) and Shigatoxin-producing Escherichia coli (STEC) share the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains.ResultsOf 51 eae positive bovine E. coli strains, 59% were classified as EPEC and 41% as STEC. EPEC strains belonged to 18 O:H serotypes, six strains to typical EPEC serogroups. EPEC strains harbored a variety of intimin variants with eae-β1 being most frequently found. Moreover, nine EPEC strains harbored ast A (EAST1), seven bfpA (bundlin), and only one strain was positive for the EAF plasmid. We have identified a new intimin gene (η2) in three bovine bfpA and astA-positive EPEC strains of serotype ONT:H45. STEC strains belonged to seven O:H serotypes with one serotype (O103:H2) accounting for 48% of the strains. The majority of bovine STEC strains (90%) belonged to five serotypes previously reported in association with hemolytic uremic syndrom (HUS), including one O157:H7 STEC strain. STEC strains harbored four intimin variants with eae-ε1 and eae-γ1 being most frequently found. Moreover, the majority of STEC strains carried only stx 1 genes (13 strains), and was positive for ehxA (18 strains) encoding for Enterohemolysin. Four STEC strains showed a virulence pattern characteristic of highly virulent human strains (stx 2 and eae positive).ConclusionOur data confirm that ruminants are an important source of serologically and genetically diverse intimin-harboring E. coli strains. Moreover, cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of EPEC and eae positive non-O157 STEC, which are described in association with human diseases.

Cold Stress Tolerance of Listeria monocytogenes: A Review of Molecular Adaptive Mechanisms and Food Safety Implications

The foodborne pathogen Listeria monocytogenes has many physiological adaptations that enable survival under a wide range of environmental conditions. The microbes overcome various types of stress, including the cold stress associated with low temperatures in food-production and storage environments. Cold stress adaptation mechanisms are therefore an important attribute of L. monocytogenes, enabling these food pathogens to survive and proliferate to reach minimal infectious levels on refrigerated foods. This phenomenon is a function of many molecular adaptation mechanisms. Therefore, an improved understanding of how cold stress is sensed and adaptation measures implemented by L. monocytogenes may facilitate the development of better ways of controlling these pathogens in food and related environments. Research over the past few years has highlighted some of the molecular aspects of cellular mechanisms behind cold stress adaptation in L. monocytogenes. This review provides an overview of the molecular and physiological constraints of cold stress and discusses the various cellular cold stress response mechanisms in L. monocytogenes, as well as their implications for food safety.

HIV-1 reverse transcriptase and integrase enzymes physically interact and inhibit each other

Ordered molecular interactions and structural changes must take place within the human immunodeficiency virus type 1 (HIV‐1) preintegration complex at various stages for successful viral replication. We demonstrate both physical and biochemical interactions between HIV‐1 reverse transcriptase and integrase enzymes. This interaction may have implications on the in vivo functions of the two enzymes within the HIV‐1 replication complex. It may be one of the various molecular interactions, which facilitate efficient HIV‐1 replication within the target cells.

The Contribution of Transcriptomic and Proteomic Analysis in Elucidating Stress Adaptation Responses of Listeria monocytogenes

The foodborne transmission of Listeria monocytogenes requires physiological adaptation to various conditions, including the cold, osmotic, heat, acid, alkaline, and oxidative stresses, associated with food hygiene, processing, and preservation measures. We review the current knowledge on the molecular stress adaptation responses in L. monocytogenes cells as revealed through transcriptome, proteome, genetic, and physiological analysis. The adaptation of L. monocytogenes to stress exposure is achieved through global expression changes in a large number of cellular components. In addition, the cross-protection of L. monocytogenes exposed to different stress environments might be conferred through various cellular machineries that seem to be commonly activated by the different stresses. To assist in designing L. monocytogenes mitigation strategies for ready-to-eat food products, further experiments are warranted to specifically evaluate the effects of food composition, additives, preservatives, and processing technologies on the modulation of L. monocytogenes cellular components in response to specific stresses.

The alternative sigma factor σL of L. monocytogenes promotes growth under diverse environmental stresses

Listeria monocytogenes are important foodborne pathogens that can cause outbreaks of serious human disease. These organisms frequently colonize and proliferate on preserved food products despite exposure to stress conditions induced by low storage temperatures, inclusion of organic acid-based preservatives, and high osmolarity. To assess alternative sigma factor sigma(L) contributions to such stress resistance of L. monocytogenes, quantitative RT-PCR assays and sigL gene deletion mutagenesis were applied in L. monocytogenes EGDe. Transcription of sigL was significantly induced by growth of EGDe under cold, organic acid, and elevated NaCl salt concentration stress conditions. The growth of a DeltasigL strain exposed to these stress conditions was also found to be significantly impaired in comparison to that of its isogenic wild-type strain. The contribution of sigma(L) to transcription control of cold and NaCl stress adaptation genes, oppA, cspD, and clpP, was also comparatively assessed in DeltasigL and wild-type EGDe cells. Transcription of the oppA gene, which encodes the OppA protein that also promotes L. monocytogenes cold growth, was significantly reduced in cold stress-grown DeltasigL cells compared to levels of the wild-type EGDe strain. These findings therefore suggest important roles of sigma(L) regulatory pathways in facilitating resistance of L. monocytogenes organisms against stress conditions associated with low storage temperatures, exposure to organic acid, and elevated NaCl salt concentrations.

Conventional and real-time PCR-based approaches for molecular detection and quantitation of bovine species material in edible gelatin.

The majority of edible gelatin in Europe is derived from pigskin, but a significant portion is extracted from bovine tissue. Because of the bovine spongiform encephalopathy crisis, consumers might be concerned about the gelatin used in various products. To assure consumers of the quality and safety of edible gelatin, European Union directive 1999/724/EC described general guidelines for gelatin production, including requirements for documentary proof confirming that raw materials are from animals fit for human consumption. Analytical methods to confirm gelatin documentation or raw material animal species source in the finished product are lacking. In this study, several published species-specific PCR systems were evaluated as potential molecular methods for determining the origin of the raw material used in making gelatin. A recently validated bovine species-specific PCR primer set targeting the ATPase 8 subunit gene in bovine mitochondrial DNA was suitable for detection of bovine material in gelatin. This PCR primer set was optimized using conventional and real-time PCR approaches. An evaluation of these two PCR methods confirmed the high specificity for the adopted primer set in various gelatin matrices of known origin. The inclusion of bovine gelatin in pork or fish gelatin can be detected at 0.1 to 0.001%. These PCR assays are potential molecular detection tools that can be used to routinely detect bovine gelatin either alone or as an inclusion in gelatin made from other species.

SpA, ClfA, and FnbA Genetic Variations Lead to Staphaurex Test-Negative Phenotypes in Bovine Mastitis Staphylococcus aureus Isolates

ABSTRACT Staphylococcus aureus encodes many proteins that act as virulence factors, leading to a variety of diseases, including mastitis in cows. Among these virulence factors, SpA, ClfA, ClfB, FnbA, and FnbB are important for the ability of S. aureus to adhere to and invade host cells as well as to evade host immune responses. The interaction between these S. aureus surface proteins and human immunoglobulin G and fibrinogen that are coupled to latex particles is utilized to induce latex agglutination reactions, which are used widely in diagnostic kits for confirmation of presumptive S. aureus isolates. In this study, the Staphaurex latex agglutination test was performed on a collection of confirmed bovine mastitis S. aureus isolates. Notably, 54% (43/79 isolates) of these isolates exhibited latex agglutination-negative phenotypes (Staphaurex-negative result). To gain insights into the reasons for the high frequency of Staphaurex-negative bovine mastitis S. aureus isolates, the spa, clfA, clfB, fnbA, and fnbB genes were examined. Specific genetic changes in spa, clfA, and fnbA, as well as a loss of fnbB, which may impair SpA, ClfA, FnbA, and FnbB functions in latex agglutination reactions, were detected in Staphaurex-negative S. aureus isolates. The genetic changes included a premature stop codon in the spa gene, leading to a truncated SpA protein that is unable to participate in S. aureus cell-mediated agglutination of latex particles. In addition, clfA and fnbA genetic polymorphisms were detected that were linked to ClfA and FnbA amino acid changes that may significantly reduce fibrinogen-binding activity. The genetic variations in these S. aureus isolates might also have implications for their bovine mastitis virulence capacity.

Evaluation of cold growth and related gene transcription responses associated with Listeria monocytogenes strains of different origins

The cold growth phenotypes and transcriptional activation of cold stress adaptation genes was evaluated amongst Listeria monocytogenes strains from human listeriosis cases, food products and associated production environments. Significant cold growth phenotypic variation was observed during growth of such strains in rich (BHI) as well as chemically defined minimal (MDM) nutrient conditions. While all twenty analyzed strains grew in BHI at 4 degrees C, only eight of these strains, mostly those recovered from human listeriosis cases, were also able to grow in MDM under similar cold stress. The cold growth phenotypes observed in BHI were used to define two categories of five strains each, which either displayed enhanced and poor cold tolerance relative to the rest of the strain collection. The first group (GP1) consisted of strains characterized by short lag times, whilst the second group (GP2) comprised of strains displaying prolonged lag times before growth resumption during incubation in BHI cultures at 4 degrees C. Transcription level activation of sigB, cspA and pgpH gene expression associated with cold stress exposure in a selection of GP1 and GP2 strains was assessed. Despite similar cold dependent sigB transcript induction between these two strain groups, there were significant differences observed in cold stress dependent induction of cspA and pgpH transcripts. Cold tolerant GP1 strains displayed relatively higher transcriptional activation of cspA and pgpH after cold stress exposure compared to the cold sensitive GP2 strains. This study highlights strain variability in cold stress tolerance phenotypes, as well as in strain capacity to activate specific cold adaptation gene expression responses. In addition the study also shows that enhanced and poor cold growth phenotypes are associated with particular strain capacity to activate important cold stress gene expression responses upon transition of L. monocytogenes into low temperature environments.